Extended Data Fig. 6: Changes in crystal-to-cytosolic phase transitions of PPO2 during 24-h hypoxia.

(a) Illustration of the phase transition of PPO2 under hypoxia. (b) Representative images of circulating hemocytes with PPO2⁺ crystal cells at each time point. Red arrows indicate crystalline PPO2, and green arrows indicate the cytosolic form of PPO2 (PPO2⁺ crystal cells, red; DAPI, blue). In 21% O₂, most PPO2⁺ cells contained PPO2 in its crystalline state (left). During 4 h in hypoxia, the majority of PPO2⁺ cells contained PPO2 in its cytosolic form (middle). During 7 h in hypoxia, crystal cells contained smaller crystals of PPO2 (right). (c) Crystal-to-cytosolic ratio of PPO2 protein in crystal cells changed during hypoxia up to 24 h. Quantification of PPO2 protein distribution patterns (cytosolic PPO2, green; crystalline PPO2, magenta) at each time point. d–e. The level of CAH2 mRNA was induced by 3-h hypoxia. (d) Relative mRNA expression levels of CAH2 in normoxia and 3-h hypoxia. (e) Visualization of CAH2 mRNA by SABER-FISH. lz⁺ crystal cells (lz, green) already exhibited higher CAH2 levels (CAH2, magenta; dotted yellow circle) than plasmatocytes (DAPI⁺) in normoxia, which was further enhanced by hypoxia. Quantification of CAH2 levels is shown on the right. (f) Haemolymph pH levels remained relatively constant in hypoxia. Synchronized larvae (w¹¹¹⁸) were reared in 21% O₂ and transferred to hypoxia for the indicated amounts of time, and haemolymph was extracted at 120-h AEL. (g) Three-dimensional reconstruction of PPO2 protein phases (PPO2⁺ crystal cells, yellow; DAPI, blue) at 21% O₂ (crystalline PPO2, left), in 4 h of culture in hypoxia (cytosolic PPO2, middle), or 5 h of culture in hypoxia (fractionated crystalline PPO2, right). (h) Recrystallization reduced the average volume of PPO2⁺ crystals per one crystal cell following 5-h hypoxia compared with intact PPO2 crystals generated in controls. (i) Quantification of crystalline or cytosolic PPO2 in the circulation. The numbers of cytosolic PPO2 (green) and crystalline PPO2 (magenta) gradually decreased until 4-h hypoxia. (j) Quantification of crystal cells containing crystalline or cytosolic PPO2 in the hematopoietic pocket (sessile) during 4 h of hypoxia. ΔCytosolic PPO2 indicates changes in the number of PPO2^cytosol per one larva (light blue, Y axis on the right). (k) The ratios of crystal cell sessility classified based on the PPO2 protein status shown in Fig. 4g. l–m. Oxygen binding capacity of PPO2 is critical for the hemocyte movement during hypoxia. Reintroduction of PPO2^WT into the PPO2^Δ mutant rescued hemocyte movement during 4-h hypoxia, while PPO2^H369N did not (PPO2^Δ; Notch-Gal4 UAS-PPO2^WT or UAS-PPO2^H369N). Quantification of lz⁺ crystal cells (l) or Pxn⁺ plasmatocytes (m). (n) Expression of crystal-cell-specific nlsTimer in controls (Notch-Gal4 UAS-nlsTimer) or in PPO2^Δ mutants (PPO2^Δ; Notch-Gal4 UAS-nlsTimer) (PPO2⁺ crystal cells, magenta; nlsTimer, red/green merged) in the circulation (top) or in the hematopoietic pocket (bottom) in normoxic or hypoxic conditions, corresponding to Fig. 4h. o–p. Plasmatocytes exhibited higher magenta-to-green nlsTimer ratios in the hematopoietic pocket than in the circulation (Hml^Δ-Gal4 UAS-nlsTimer). (o) Maturation of plasmatocyte-specific nlsTimer (magenta-to-green merged) in circulating cells (top) or sessile cells (bottom) at 21% O₂ (left) or 5% O₂ (right). (p) Quantification of the magenta/green ratio for each condition corresponding to panel (o). Red fluorescence of nlsTimer was converted into magenta to avoid the red/green colour scheme. (q) Illustration of the PPO2 crystal-to-cytosol transition upon hypoxia. Scale bar, 10 µm. n.s (not significant), **P < 0.01, ***P < 0.001, ****P < 0.0001. The single red dots in Extended Data Fig. 6c indicate the percentage of crystal cells with PPO2^crystal per one larva. Extended Data Fig. 6c was analysed by one-way ANOVA followed by Tukey’s post hoc test. Extended Data Fig. 6d, e, h, i, and p were analysed using the Mann–Whitney test. Extended Data Fig. 6l and m was analysed by two-way ANOVA followed by Dunnett’s multiple comparison test. Box and whiskers plots in 6c, 6e, 6h, 6l, 6m, and 6p denote maximum, 25%, median, 75%, and minimum values, respectively. Error bars in 6d, 6i, and 6j represent standard deviations (SDs). Schematic diagrams in Extended Data Fig. 6q was created with BioRender.com.

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